A method for regulating oxygen stress in Saccharomyces cerevisiae using Y-family polymerase rev1

A fermentation technology of Saccharomyces cerevisiae and yeast, applied in the field of bioengineering, can solve problems such as protein oxidative damage, yeast toxicity, and increased ROS level, and achieve the effect of enhancing oxygen stress resistance

Active Publication Date: 2021-07-27
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, during the fermentation process, the concentration of pyruvate is too high, which is toxic to the yeast, and the high acid concentration will lead to an increase in the level of intracellular ROS, causing oxidative damage to DNA and proteins.

Method used

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  • A method for regulating oxygen stress in Saccharomyces cerevisiae using Y-family polymerase rev1
  • A method for regulating oxygen stress in Saccharomyces cerevisiae using Y-family polymerase rev1
  • A method for regulating oxygen stress in Saccharomyces cerevisiae using Y-family polymerase rev1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of deletion mutant strains

[0037] Using the wild-type Saccharomyces cerevisiae genome as a template and P1 / P2, P3 / P4, and P5 / P6 as primers, the left arm (L), histidine gene (M) and right arm of the REV1 gene to be knocked out were amplified. (R), the knockout box REV1-LMR was constructed by fusion PCR ( figure 1 ). The correctly sequenced knockout box was introduced into the starting strain Saccharomyces cerevisiae BY4741 by chemical transformation, the positive transformants were screened by histidine marker gene, and the genome was extracted and verified by PCR sequencing.

[0038] Table 1 Primer sequence list

[0039]

Embodiment 2

[0040] Example 2: Construction of overexpression strains

[0041] Using the genome of Saccharomyces cerevisiae BY4741 as the template and P7 / P8 as the primers to amplify the target gene REV1, the amplified product was digested with the same restriction enzymes EcoRI and XhoI as the plasmid PY26, and the gene REV1 was ligated to PY26 by T4 ligase , the strong promoter GPD1 initiates transcription and translation, and the URA3 gene on the recombinant plasmid is used to screen positive transformants. Finally, the plasmid is extracted to verify that the overexpression strain Rev1Δ / REV1 is obtained.

Embodiment 3

[0042] Example 3: Determination of growth performance of each strain

[0043] (1) Plate growth experiment: A single colony of the strain to be tested was inoculated into 20 mL of YNB (0.67% YeastNitrogen Base without Amino Acids, 2% Glucose) liquid medium for overnight activation, and then transferred to YNB medium and cultured until the For several periods, determine the bacterial concentration and adjust the bacterial suspension to OD 600 = 1.0, take this as the initial concentration, carry out 5 times of 10-fold gradient dilution, sequentially inoculate 3 μL of bacterial liquid on the corresponding solid YNB medium, cultivate at 30 ° C for 2-3 days, observe the growth of the bacterial cells and take pictures ( figure 2 ).

[0044] (2) Growth curve test: Inoculate a single colony of the strain to be tested in 20 mL of YNB (0.67% YeastNitrogen Base without Amino Acids, 2% Glucose) liquid medium for overnight activation, and then transfer to the corresponding YNB liquid medi...

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Abstract

The invention discloses a method for regulating oxygen stress of Saccharomyces cerevisiae by using Y family polymerase Rev1, and belongs to the technical field of biological engineering. The gene REV1 was knocked out by homologous recombination, and the knockout strain rev1Δ was constructed, which reduced the ability of the yeast strain to resist oxidative stress. Specifically, the biomass of the knockout strain was reduced by 9% under stress conditions, and the survival rate was reduced by 34.8%. The frequency of gene mutation decreased by 63.8%, and pyruvate production decreased by 30.8%. The pY26 plasmid was used to overexpress the gene REV1, and the overexpression strain rev1Δ / REV1 was constructed, which enhanced the ability of the strain to resist oxidative stress. The specific performance was that the biomass increased by 8.3%, the survival rate increased by 11.5%, the gene mutation frequency increased by 186.2%, and the pyruvate production increased. 57.7%. The above results indicated that Rev1 could improve the viability and fermentation performance of yeast strains under oxygen stress conditions.

Description

technical field [0001] The invention relates to a method for regulating oxygen stress of Saccharomyces cerevisiae by using Y family polymerase Rev1, and belongs to the technical field of bioengineering. Background technique [0002] Microorganisms are easily attacked by various exogenous and endogenous environments in the process of growth and metabolism. The exogenous environment mainly includes ionizing radiation, ultraviolet radiation and various chemical reagents, and the endogenous environment mainly includes some secondary substances produced by cells in the process of metabolism. grade metabolites. Most of these factors can cause the accumulation of reactive oxygen species in cells, causing oxidative damage to intracellular DNA and hindering cell growth and metabolism. In industrial applications, Saccharomyces cerevisiae can be used not only to produce alcohol, but also to produce organic acids, proteins, etc. In the field of life sciences, Saccharomyces cerevisiae ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/40C12R1/865
CPCC12N9/1241C12N15/81C12P7/40
Inventor 吴静姚瑞刘立明陈修来刘佳罗秋玲
Owner JIANGNAN UNIV
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